Journal: eLife

Article Title: High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen

doi: 10.7554/eLife.87496

Figure Lengend Snippet: The C57BL/6 mice were treated with NN and HH for 7 days, and the spleen was collected for subsequent detection. ( A ) Western blot detection of HO-1, Ft-L, Ft-H, NCOA4, Fpn and TfR protein expression in spleen. ( B ) Statistical analysis of HO-1, Ft-L, Ft-H, NCOA4, Fpn, and TfR protein expression. ( C ) GEO data analysis of HMOX1, FTL, FTH1, NCOA4, SLC40A1, and TFRC mRNA expression in PMBCs before and after climbing to HA (n=98). ( D ) Western blot analysis for ACSL4, GPX4, and xCT protein expression in the spleen, with ( E ) depicting the statistical analysis of these protein levels. ( F ) GEO data analysis of ACSL4, GPX4 and SLC7A11 mRNA expression in PMBCs before and after reaching HA (n=98). ( G ) The content of Fe 2+ in the spleen was detected using the FerroFarRed probe by flow cytometry. ( H ) The level of lipid ROS in the spleen was detected using the C11-BODIPY probe by flow cytometry. The MDA ( I ), Cys ( J ) and GSH ( K ) levels in the spleen were detected using biochemical detection kits, respectively. ( L ) Lillie staining of Fe 2+ in the spleen after 7 days of HH exposure. ( M ) Fe 2+ deposition in the spleen as described in ( L ) was quantified. Data are expressed as the means ± SEM (n=3 per group); * p<0.05, ** p<0.01, *** p<0.001 versus the NN group or the indicated group. Figure 8—source data 1. PDF containing and original scans of the relevant western blot analysis (anti-HO-1, anti-Ft-L, anti-Ft-H, anti-NCOA4, anti-Fpn, anti-TfR and anti-β-actin) with highlighted bands and sample labels. Figure 8—source data 2. PDF containing and original scans of the relevant western blot analysis (anti-ACSL4, anti-GPX4, anti-xCT and anti-β-actin) with highlighted bands and sample labels.

Article Snippet: The membranes were incubated with the following antibodies: HO-1 (1:2000, Abcam, ab13243, USA); Ft-L (1:1000, Abcam, ab69090, USA); Ft-H (1:1000, Novus, NBP1-31944, USA); NCOA4 (1:1000, Santa Cruz, sc-373739, USA); TfR (1:1000, Thermo Fisher, 13–6800, USA); Fpn (1:1000, Novus, NBP1-21502, USA); ACSL4 (1:1000, Santa Cruz, sc-271800, USA); xCT (1:1000, Proteintech, 26864–1-AP, USA); Gpx4 (1:1000, Abcam, ab125066, USA); CD206 (1:1000, RD, AF2535, USA); and β-actin (1:1000, Sigma‒Aldrich, A5316, USA).

Techniques: Western Blot, Expressing, Flow Cytometry, Staining